Characterization of flavin-based fluorescent proteins: an emerging class of fluorescent reporters.
Characterization of flavin-based fluorescent proteins: an emerging class of fluorescent reporters.
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DOI：
10.1371/journal.pone.0064753
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发表时间：
2013
期刊：
影响因子：
3.7
通讯作者：
Schroeder CM
中科院分区：
文献类型：
--
作者：
Mukherjee A;Walker J;Weyant KB;Schroeder CM
基于黄素结合光传感器的荧光报告蛋白是近年来发展起来的一类新型基因编码探针，其特点是体积小，荧光成熟不依赖于氧。基于黄素的荧光蛋白（FbFPs）解决了现有绿色荧光蛋白（GFP）衍生的荧光报告的两个主要限制，即GFP的荧光总体上大尺寸和氧依赖性成熟。然而，FbFPs正处于发展的初级阶段，仅在少数生物学研究中得到利用。重要的是，缺乏对FbFPs作为一套实用生物探针的性能和特性的充分理解。在这项工作中，我们广泛地表征了从恶臭假单胞菌、枯草芽孢杆菌和拟南芥中分离的三种FbFPs，使用体外研究来评估探针亮度、寡聚物状态、成熟时间、荧光全蛋白的比例、pH耐受性、氧化还原敏感性和热稳定性。此外，我们通过构建一系列基于fbfp的转录构建物来探测大肠杆菌启动子活性，验证了FbFPs是稳定的分子标签。总体而言，FbFPs作为广谱生物报告者具有关键优势，包括强大的pH耐受性（4-11）、热稳定性（高达60°C）和荧光快速成熟（<3分钟）。此外，来自拟南芥（iLOV）的FbFP成为大肠杆菌启动子活性的稳定且无扰动的报告基因。我们的研究结果表明，基于fbfp的报告具有解决与使用GFP相关的关键限制的潜力，例如ph敏感荧光和荧光成熟缓慢的动力学（10-40分钟为最大荧光恢复的一半）。从这个角度来看，fbfp代表了荧光报告蛋白调色板的一个有用的新成员，我们的研究结果构成了一个重要的框架，使研究人员能够实现和进一步设计改进的基于fbfp的报告蛋白，其亮度增强，黄素结合更紧密，这将最大化其潜在的好处。
Fluorescent reporter proteins based on flavin-binding photosensors were recently developed as a new class of genetically encoded probes characterized by small size and oxygen-independent maturation of fluorescence. Flavin-based fluorescent proteins (FbFPs) address two major limitations associated with existing fluorescent reporters derived from the green fluorescent protein (GFP)–namely, the overall large size and oxygen-dependent maturation of fluorescence of GFP. However, FbFPs are at a nascent stage of development and have been utilized in only a handful of biological studies. Importantly, a full understanding of the performance and properties of FbFPs as a practical set of biological probes is lacking. In this work, we extensively characterize three FbFPs isolated from Pseudomonas putida, Bacillus subtilis, and Arabidopsis thaliana, using in vitro studies to assess probe brightness, oligomeric state, maturation time, fraction of fluorescent holoprotein, pH tolerance, redox sensitivity, and thermal stability. Furthermore, we validate FbFPs as stable molecular tags using in vivo studies by constructing a series of FbFP-based transcriptional constructs to probe promoter activity in Escherichia coli. Overall, FbFPs show key advantages as broad-spectrum biological reporters including robust pH tolerance (4–11), thermal stability (up to 60°C), and rapid maturation of fluorescence (<3 min.). In addition, the FbFP derived from Arabidopsis thaliana (iLOV) emerged as a stable and nonperturbative reporter of promoter activity in Escherichia coli. Our results demonstrate that FbFP-based reporters have the potential to address key limitations associated with the use of GFP, such as pH-sensitive fluorescence and slow kinetics of fluorescence maturation (10–40 minutes for half maximal fluorescence recovery). From this view, FbFPs represent a useful new addition to the fluorescent reporter protein palette, and our results constitute an important framework to enable researchers to implement and further engineer improved FbFP-based reporters with enhanced brightness and tighter flavin binding, which will maximize their potential benefits.
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影响因子：
3.7
作者：
Geiger K;Leiherer A;Muendlein A;Stark N;Geller-Rhomberg S;Saely CH;Wabitsch M;Fraunberger P;Drexel H
通讯作者：
Drexel H
影响因子：
7.4
作者：
Kasahara, M;Swartz, TE;Briggs, WR
通讯作者：
Briggs, WR
DOI：
10.1038/nrmicro2622
发表时间：
2011-08-08
期刊：
Nature reviews. Microbiology
影响因子：
--
作者：
通讯作者：
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在研究宿主 - 微生物相互作用中，底部厌氧菌的遗传转化，用于FMN绿色荧光蛋白表达。
影响因子：
3.7
作者：
Choi CH;DeGuzman JV;Lamont RJ;Yilmaz Ö
通讯作者：
Yilmaz Ö
影响因子：
13.8
作者：
CUBITT, AB;HEIM, R;TSIEN, RY
通讯作者：
TSIEN, RY